WO2013159227A1 - Mutants constitutivement actifs des récepteurs du goût amer - Google Patents

Mutants constitutivement actifs des récepteurs du goût amer Download PDF

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WO2013159227A1
WO2013159227A1 PCT/CA2013/050313 CA2013050313W WO2013159227A1 WO 2013159227 A1 WO2013159227 A1 WO 2013159227A1 CA 2013050313 W CA2013050313 W CA 2013050313W WO 2013159227 A1 WO2013159227 A1 WO 2013159227A1
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receptor
cam
amino acid
wild type
alanine
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Prashen CHELIKANI
Sai PYDI
Rajinder P:. BHULLAR
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University Of Manitoba
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the human taste perception is one of the most important chemosensations. Humans can sense five basic tastes which are sweet, bitter, umami, salt and sour. Bitter taste which is sensed by bitter taste receptors (T2Rs) is the most complex and the least understood among the human taste sensations, and part of this complexity is due to the large number of receptors (25 T2Rs) that code for bitter taste sensation compared to only three receptors that code for both sweet and umami tastes. Recent studies have shown that T2Rs are expressed in many extra-oral tissues including the brain (Singh ei al. 2011 b), where their physiological function needs to be determined. The T2Rs are cell surface receptors and belong to the G-protein coupled receptor (GPCR) superfamily (Chandrashekar et al. 2000).
  • GPCR G-protein coupled receptor
  • the amino acid numbering used in this manuscript incorporates the residue number from the receptor sequence (e.g. Gly28) and a residue number (e.g. 1.46) from a generic numbering system developed by Ballesteros and Weinstein (Ballesteros & Weinstein 1995).
  • the two residues at positions 1.46 and 7.47 are 88% and 70% conserved in T2Rs (Singh ei al. 201 1a), however only glycine at position 1.46 is conserved in Class A GPCRs (Smith 2010, Singh et al. 201 1a).
  • Gly51 1,46 occurs as naturally occurring polymorphic variants G51A, G51V, and G51 D which cause autosomal dominant retinitis pigmentosa (ADRP) (Sung et al. 1991 , Dryja et al. 1991 , Macke et al. 1993). Although these ADRP mutants fold properly, they displayed thermally destabilized structures and were severely defective in signal transduction (Bosch-Presegue et al. 2011 , Bosch et al. 2003).
  • ADRP autosomal dominant retinitis pigmentosa
  • an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) selected from the group consisting of:
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to histidine 214 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3,
  • T2R CAM receptor selected from the group consisting of: T2R4,
  • T2R13 and T2R41 wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to glutamine 216 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to valine 234 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R4, T2R10 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to methionine 237 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60, wherein said T2R CAM receptor further comprises a alanine residue at the amino acid residue corresponding to histidine 123 of the T2R4 wild type sequence;
  • T2R4 CAM receptor wherein said T2R4 mutant receptor further comprises an alanine residue at amino acid residue corresponding to asparagine 132 of the T2R4 wild type sequence.
  • an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) selected from the group consisting of:
  • a method of preparing a constitutively active mutant (CAM) bitter taste receptor comprising: a) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, to site directed mutagenesis such that the histidine residue corresponding to histidine 214 is mutated to alanine; or
  • T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39,
  • T2R40, T2R48, T2R49, T2R50 and T2R55 to site directed mutagenesis such that the serine residue corresponding to serine 285 of the T2R4 wild type sequence is mutated to alanine ;
  • T2R receptor selected from the group consisting of: T2R4, T2R13 and T2R41 to site directed mutagenesis such that the glutamine residue corresponding to glutamine 216 of the T2R4 wild type sequence is mutated to alanine; or
  • T2R receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10 to site directed mutagenesis such that the valine residue corresponding to valine 234 of the T2R4 wild type sequence is mutated to alanine; or e) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R10 and T2R55 to site directed mutagenesis such that the methionine residue corresponding to methionine 237 of the T2R4 wild type sequence is mutated to alanine; or
  • T2R receptor selected from the group consisting of: T2R3,
  • T2R receptor selected from the group consisting of: T2R1 ,
  • T2R4 receptor subjecting a T2R4 receptor to site directed mutagenesis such that asparagine residue 132 is mutated to alanine; and recovering a nucleic acid molecule encoding the T2R4 CAM receptor.
  • a method of determining if a compound of interest is a human bitter taste receptor blocker comprising:
  • the compound of interest is a bitter taste receptor antagonist or inverse agonist if said compound reduces the activity of the constitutively active bitter taste receptor mutant to approximately the same activity as a wild type bitter taste receptor.
  • a nucleic acid molecule encoding a bitter taste receptor (T2R) constitutively active mutant (CAM) comprising a nucleotide sequence encoding the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9 or as described above.
  • T2R bitter taste receptor
  • CAM constitutively active mutant
  • FIG. 1 Two-dimensional representation of the T2R4 amino acid sequence with the octapeptide FLAG-tag at the N-terminus.
  • the coding region of T2R4 without the FLAG-tag is 299 amino acids.
  • the receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three intracellular loops (ICLs), and a cytoplasmic tail which constitutes a short helix-8 that runs parallel to the membrane.
  • TM transmembrane
  • ECLs extracellular loops
  • ICLs intracellular loops
  • T2R1 SEQ ID NOs: 10 and 11
  • T2R4 SEQ ID NOs: 12 and 13
  • opsin SEQ ID NOs: 14 and 15
  • FIG. 1 Characterization of Ga16/44 chimera-mediated signaling of the wild type T2R4 and mutants.
  • FIG. 1 Pharmacological characterization of WT-T2R4 and mutants. Shown are the activity after stimulation (A, Top panel) with a single saturating concentration
  • Rhodopsin_1 U19 represents the inactive structure of rhodopsin (Protein Data Bank code 1 U19) and CAM_Rhodopsin_2X72 represent the structure of the constitutively active mutant rhodopsin (Protein Data Bank code 2X72).
  • T2R4_basal represents the inactive structure of wild type T2R4 built by homology modeling using the Rhodopsin_1 U19 template
  • T2R4_S285A 30 mutant represents the constitutively active S285A mutant built by homology modeling using the CAM_Rhodopsin_2X72 template.
  • T2R4 basal model an intrahelical hydrogen bond connects the side chains of S285 7 47 , R63 2 54 with Water 2017, and interhelical backbone contacts between N32 1 ' 50 and G28 1 ,46 are observed. There is a rearrangement of this network in the constitutively active S285A mutant, due to loss of hydrogen bonding by the residue at position 7.47.
  • FIG. 6 Comparison of the hydrogen-bond network in the vicinity of residues 1.46 and 7.47, in a molecular model of S285P mutant.
  • the S285P model was built by homology modeling using the rhodopsin_1 U19 as template.
  • the interhelical H-bond interaction between the backbone N-atom of Pro285 7 ' 47 with the side chain-NH2 of Arg63 254 restrains basal activity of the receptor. This is similar to the H-bond interaction of Ser285 7 47 with Arg63 2 54 observed in the molecular model of wild type T2R4 that is proposed to stabilize the inactive state.
  • Broken blue lines represent hydrogen bonds.
  • FIG. 8 Two-dimensional representation of the T2R4 amino acid sequence with the FLAG-tag at the N-terminus.
  • the receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three
  • ICLs intracellular loops
  • cytoplasmic tail The 23 ICL3 residues mutated to alanine in this study are displayed in broken rings. The constitutively active mutants identified in this study are represented in grey circles.
  • FIG. 9 Representative calcium traces for HEK293T cells transiently transfected with T2R4 and select mutants.
  • the mock transfected (pcDNA) control is shown.
  • the cells are stimulated with 2.5 mM quinine (top panel) or assay buffer (lower panel).
  • the calcium mobilized (ARFUs or Relative Fluorescence Units) was detected using the calcium sensitive dye Fluo 4NW (Invitrogen), and fluorescence measured using Flex Station III microplate reader.
  • FIG. 10 A. Pharmacological characterization of the basal or agonist independent activity of WT-T2R4 and intracellular alanine mutants. Calcium mobilized (ARFU) is normalized to WT-T2R4 cell surface expression as determined by ELISA. The results were analyzed using one way ANOVA with Tukeys post hoc test, at significance level p ⁇ 0.05.
  • FIG. 11 Homology models of the inactive (red) and constitutively active (yellow) WT-T2R4 built using the rhodopsin inactive (PDB: 1 U19) and CAM (PDB: 2X72) structures as templates.
  • A. The left panel shows the intracellular view (from the cytoplasmic side) of the TM2-TM3-TM5-TM6 arrangement in both T2R4 structures. The intracellular loops (ICLs) are shown as threads, along with the location of the ICL3 CAMs.
  • B. The right panel shows the membrane view of TM5-TM6, along with ICL3 CAMs and the packing interactions of the LxxSL motif on TM5. In the T2R4 CAM model, the cytoplasmic end of TM6 moves by around 2A towards the helical core.
  • FIG. 12 Two-dimensional representation of the T2R4 amino acid sequence with the FLAG-tag at the N-terminus.
  • the receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three
  • ICLs intracellular loops
  • cytoplasmic tail The intracellular residues mutated to alanine in this study, are displayed in broken rings, the constitutively active mutants are represented in grey circles, and the short helix in ICL2 Is shown.
  • FIG. 13 A. Pharmacological characterization of the basal or agonist independent activity of WT-T2R4 and intracellular alanine mutants. Calcium mobilized (ARFU) is normalized to WT-T2R4 cell surface expression as determined by ELISA. The results were analyzed using one way ANOVA with Tukeys post hoc test, at significance level p ⁇ 0.05.
  • CAM T2R constitutively active mutants
  • T2R receptors there are at least 25 known T2R receptors. Accordingly and as discussed below, T2R receptors in which the above-referenced amino acids are conserved when compared to the T2R4 sequence can be used in the generation of corresponding CAMs. For example any T2R receptor having a serine residue at the residue corresponding to serine 285 in T2R4 can be mutated at that residue to alanine and a CAM T2R receptor will be obtained.
  • the appropriate T2Rs for each of the 8 CAMs are listed below.
  • CAM T2R receptor comprising or having the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.
  • a purified or isolated CAM T2R receptor comprising or having the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO:3.
  • S285A mutant displayed agonist- independent or constitutive activity, while the conservative replacement S285T displayed wild type basal activity.
  • Ser285 747 stabilizes the inactive state of T2R4 by forming a hydrogen bond with Arg63 254 .
  • the constitutive activity of the S285A mutant is due to a loss in ability of the residue at position 7.47 in T2R4 to hydrogen bond. This leads to a re-arrangement of the hydrogen-bond network connecting TM1-TM2- TM7 causing the S285A mutant to adopt an active conformation.
  • no major changes in T2R function were observed upon mutation of the glycine at position 1.46.
  • the inventors have carried out alanine scan mutagenesis of the ICL3 and functionally characterized 23 alanine mutants of T2R4.
  • the results based on site-directed mutagenesis, pharmacological characterization of the mutants, and molecular modeling analysis allowed the inventors to identify four constitutively active mutants (CAMs) in ICL3, with constitutive activity ranging from 2 to 10 fold over wild type T2R4.
  • CAMs constitutively active mutants
  • T2Rs bitter taste receptors
  • T2R agonists also known as T2R agonists
  • T2R agonists have diverse chemical structures and include plant derived compounds and natural alkaloids such as, quinine, caffeine, nicotine and morphine.
  • bitter blockers antagonists and/or inverse agonists
  • CAMs are mutations in the receptor that lock it in an active conformation and allow the receptor to signal, even in the absence of an agonist.
  • 17 have the amino acid serine in transmembrane helix 7 at position 7.47 (according to Ballesteros and Weinstein numbering) or corresponding to serine 285 in the T2R4 wild type sequence (SEQ ID NO:1 ).
  • T2Rs Replacement of the serine at position 7.47 in T2Rs with alanine results in a CAM phenotype, as observed in the case of serine to alanine mutation at 7.47 in T2R4.
  • the CAMs of T2Rs can be used as pharmacological tools for the screening of potential bitter blockers.
  • a purified or isolated T2R CAM in which position 7.47 or amino acid 285 is an alanine wherein the corresponding wild type sequence has a serine at position 7.47 or amino acid 285 of the T2R4 wild type sequence.
  • the wild type T2R having serine at amino acid 285 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55.
  • the T2R having a serine at amino acid position 285 of the wild type sequence is T2R4.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to 214 is an alanine wherein the corresponding wild type sequence has a histidine at position 214 of the wild type T2R4 sequence.
  • the wild type T2R having histidine at amino acid 214 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2 . R47, T2R48, T2R49, T2R50, T2R55 and T2R60.
  • the T2R having a histidine at amino acid position 2 4 of the wild type sequence is T2R4.
  • T2R4, T2R13 and T2R41 are examples of T2R4, T2R13 and T2R41 .
  • a purified or isolated T2R CAM in which amino acid 216 is an alanine wherein the corresponding wild type T2R4 sequence has a glutamine at this position.
  • the wild type T2R having glutamine at amino acid 216 is selected from the group consisting of: T2R4, T2R13 and T2R41.
  • the T2R having a glutamine at amino acid position 216 of the wild type sequence is T2R4.
  • T2Rs in humans 4 have the amino acid valine at amino acid residue 234 of the wild type T2R4 sequence: T2R4, T2R7, T2R8 and T2R 0.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to 234 is an alanine wherein the corresponding wild type T2R4 sequence has a valine at position 234.
  • the wild type T2R having valine at amino acid 234 is selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10.
  • the T2R having a valine at amino acid position 234 of the wild type sequence is T2R4.
  • T2Rs Of the 25 T2Rs in humans, 3have the amino acid methionine at amino acid residue 237: T2R4, T2R10 and T2R55.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to 237 is an alanine wherein the corresponding wild type T2R4 sequence has a methionine at position 237.
  • the wild type T2R having methionine at amino acid 237 is selected from the group consisting of: T2R4, T2R10 and T2R55. -Preferably, the T2R having a methionine at amino acid position 237 of the wild type sequence is T2R4.
  • T2Rs in humans 21 have the amino acid isoleucine at amino acid residue 55: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R14, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to position 55 is an alanine wherein the corresponding wild type T2R4 sequence has an isoleucine at position 55.
  • the wild type T2R having isoleucine at amino acid 55 is selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55.
  • the T2R having a methionine at amino acid position 237 of the wild type sequence is T2R4.
  • T2Rs Of the 25 T2Rs in humans, 12 have the amino acid histidine at amino acid residue 123:T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to 123 is an alanine wherein the corresponding wild type T2R4 sequence has a histidine at position 123.
  • the wild type T2R having histidine at amino acid 123 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60.
  • the T2R having a methionine at amino acid position 123 of the wild type sequence is T2R4.
  • T2Rs Of the 25 T2Rs in humans, 1 has the amino acid asparagine at amino acid residue 132: T2R4.
  • a purified or isolated T2R CAM in which amino acid residue corresponding to 132 is an alanine wherein the corresponding wild type T2R4 sequence has an asparagine at position 132.
  • the T2R having an asparagine at amino acid position 132 of the wild type sequence is T2R4.
  • the sequence of the human T2R4 wild type receptor is provided below.
  • T2R4 S285A CAM The sequence of the T2R4 S285A CAM is provided below.
  • the sequence of the human T2R4 H214A CAM is provided below.
  • T2R4 H214A CAM sequence (SEQ ID No. 3)
  • T2R4 Q216A CAM receptor The sequence of the T2R4 Q216A CAM receptor is provided below.
  • T2R4 Q216A CAM sequence (SEQ ID No. 4)
  • T2R4 V234A CAM receptor The sequence of the T2R4 V234A CAM receptor is provided below.
  • T2R4 V234A CAM sequence (SEQ ID No. 5)
  • T2R4 M237A CAM receptor The sequence of the T2R4 M237A CAM receptor is provided below.
  • T2R4 M237A CAM sequence (SEQ ID No. 6)
  • T2R4 I55A CAM receptor The sequence of the human T2R4 I55A CAM receptor is provided below.
  • T2R4 I55A CAM sequence SEQ ID No. 7
  • T2R4 H123A CAM receptor The sequence of the T2R4 H123A CAM receptor is provided below.
  • T2R4 H123A CAM sequence (SEQ ID No. 8)
  • T2R4 N132A CAM sequence (SEQ ID No. 9)
  • an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) mutant selected from the group consisting of:
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to histidine 214 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further
  • T2R CAM receptor selected from the group consisting of: T2R4, T2R13 and T2R41.wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to glutamine 216 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to valine 234 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R4, T2R10 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to methionine 237 of the T2R4 wild type sequence;
  • T2R CAM receptor selected from the group consisting of: T2R3, T2R4,
  • T2R CAM receptor selected from the group consisting of: T2R1 , T2R3,
  • T2R4 CAM receptor wherein said T2R4 mutant receptor further comprises an alanine residue at amino acid residue corresponding to aspargine 132 of the T2R4 wild type sequence.
  • nucleic acid molecule encoding the selected T2R is mutated through site directed
  • nucleic acid molecule encoding the CAM can then be engineered for expression in a suitable cell line, either for expression of the CAM for recovery or in a cell line for testing and/or screening, as discussed herein.
  • a method of generating or producing such a T2R CAM by following the steps as outlined above, specifically, the mutation of the selected wild type T2R nucleotide sequence at the selected amino acid residue to alanine, thereby producing the CAM.
  • a cell or cell line engineered to express the constitutively active bitter taste receptor mutant(s) described herein there is provided a cell or cell line engineered to express the constitutively active bitter taste receptor mutant(s) described herein.
  • suitable cells or cell lines for in vitro expression of the T2R CAMs which will be readily apparent to one of skill in the art.
  • Such cells or cell lines can then be used to screen for bitter blockers as antagonists or inverse agonists, as discussed herein.
  • T2R CAM comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.
  • nucleic acid molecule encoding an amino acid sequence that is a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.
  • T2R4 CAM peptide sequence modifications made to the T2R4 CAM peptide sequence that do not significantly affect the constitutive activity of this peptide are within the scope of the invention. For example, conservative changes within highly non-conserved amino acids are likely to be tolerated. It is of note that such conserved amino acids can be readily determined by comparison of two or more of the T2R sequences. Furthermore, the 3D structures of these receptors have been modeled and accordingly it is well within routine skill in the art to determine what amino acid locations would tolerate modification (and what modifications would be tolerated). Other such possible substitutions will be readily apparent to one of skill in the art and/or through routine experimentation.
  • T2R CAMs As will be appreciated by one of skill in the art, taste and flavour companies interested in discovering new bitter blockers can use these T2R CAMs as
  • a bitter blocker (antagonist and/or inverse agonist) is expected to decrease the constitutive activity of these CAMs, as discussed below.
  • the current technique(s) for elucidating putative bitter blockers is to use competition and/or inhibition assays where premixing of the agonist and putative antagonist is performed and the cellular response of this mixture is then compared to those of agonist responses.
  • the CAMs in T2Rs are more accurate in testing the efficacy of the putative bitter blockers.
  • putative bitter blockers should be able to reverse the basal activity of CAMs, depending on their potency.
  • Using CAMs allows for the pharmacological classification of the putative bitter blockers as antagonists or inverse agonists, depending on their ability to attenuate the basal signal to either wild type or lower than wild type levels.
  • isolated or purified does not require absolute purity but rather that the receptor is greatly enriched or overexpressed compared to its natural environment.
  • a method of determining if a compound of interest is a human bitter taste receptor blocker comprising:
  • the compound of interest is a bitter taste receptor antagonist or inverse agonist if said compound reduces the activity of the constitutively active bitter taste receptor mutant to approximately the same activity as a wild type bitter taste receptor.
  • the activity of the T2R receptor may be determined by any means known in the art, for example, by measuring intracellular Ca 2+ levels.
  • the activity of the constitutively active bitter taste receptor mutant is compared to a control.
  • the control may be a wild type bitter taste receptor.
  • the control could be a CAM T2R mutant exposed to a known negative compound (one that has no effect on CAM T2R activity) or a mock treated control.
  • the control does not necessarily need to be repeated every time. Specifically, it is believed that one of skill in the art will be able to identify whether or not a specific compound of interest without necessarily repeating controls every time.
  • bitter taste receptor agonists and reverse agonists can be identified.
  • bitter taste receptor agonist or reverse-agonist as identified by the above-described method.
  • a nucleic acid molecule encoding a human bitter taste receptor (T2R) having a serine at amino acid position 285 of the wild type sequence mutated to a non-polar amino acid.
  • T2R human bitter taste receptor
  • a nucleic acid molecule encoding a purified or isolated human bitter taste receptor (T2R) having a serine at position 7.47 of transmembrane helix 7 in the wild type receptor mutated to a non-poiar amino acid.
  • T2R human bitter taste receptor
  • nucleic acid molecule encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.
  • nucleic acid molecule encoding an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
  • nucleic acid molecules may be inserted into a suitable expression vector and transfected into a suitable cell line so that the cell line expresses CAM T2R mutants.
  • a suitable cell line can be used to screen for bitter taste receptor agonists and reverse agonists as discussed herein.
  • bitter blockers that target T2R4 and other T2Rs that have a serine at a position corresponding to amino acid 285 of T2R4 ⁇ SEQ ID NO: 1) can be characterized using the S285A T2R4 mutant as set forth in SEQ ID NO: 2.
  • bitter blockers that target T2R4 and other T2Rs that have a histidine at a position corresponding to amino acid 214 of T2R4 can be characterized using the H214A T2R4 mutant as set forth in SEQ ID NO: 3.
  • bitter blockers that target T2R4 and other T2Rs that have a glutamine at a position corresponding to amino acid 216 of T2R4 can be characterized using the Q216A T2R4 mutant as set forth in SEQ ID NO: 4.
  • bitter blockers that target T2R4 and other T2Rs that have a valine at a position corresponding to amino acid 234 of T2R4 can be characterized using the V234A T2R4 mutant as set forth in SEQ ID NO: 5.
  • bitter blockers that target T2R4 and other T2Rs that have a methionine at a position corresponding to amino acid 237 of T2R4 can be characterized using the M237A T2R4 mutant as set forth in SEQ ID NO: 6.
  • bitter blockers that target T2R4 and other T2Rs that have an isoleucine at a position corresponding to amino acid 55 of T2R4 can be characterized using the I55A T2R4 mutant as set forth in SEQ ID NO: 7.
  • bitter blockers that target T2R4 and other T2Rs that have a histidine at a position corresponding to amino acid 123 of T2R4 can be characterized using the H123A T2R4 mutant as set forth in SEQ ID NO: 8.
  • bitter blockers that target T2R4 and other T2Rs that have a aspargine at a position corresponding to amino acid 132 of T2R4 can be characterized using the N132A T2R4 mutant as set forth in SEQ ID NO: 9.
  • Bitter blockers have immense nutraceutical potential.
  • a large number of naturally occurring or plant derived compounds can be screened in a semi- or high- throughput format using these CAMs. This would allow classification of some of these putative bitter blockers into T2R antagonists and/or inverse agonists depending on their ability to attenuate the signal to varying percentages. For example, these compounds could then be screened against the US Food and Drug Administration (FDA) list of 3000 compounds in the SCOGS database classified as Generally Recognized as Safe (GRAS) for human consumption.
  • FDA US Food and Drug Administration
  • CAMs at other positions in T2Rs can be characterized using the technique described. As discussed above, an accurate estimate of basal activity is necessary for classifying a mutant as CAM although this does not necessarily need to be repeated every time. Basal activity can be calculated from slope of expression vs. basai activity for the mutants compared to same value for the wild type receptor (Hwa et al. 1997, Chakraborty et al. 2012).
  • the inventors targeted two conserved residues present on TM1 and TM7 of T2R4 for structure-function analysis. This elucidated the role of a crucial amino acid Ser285 7 ' 47 that is involved in locking T2R4 in the inactive state by interhelical hydrogen bonds.
  • the residue at position 1 .46 is conserved as a glycine in a majority of Class A GPCRs, including rhodopsin.
  • the naturally occurring variants of Gly51 1 46 cause ADRP, and like most other ADRP mutations appear to cause destabilization of the opsin structure. It was hypothesized that the instability of Gly51 1.46 ADRP mutations can be due to a steric hindrance with the residue 7.47, which is Va!300 (Bosch et al. 2003).
  • Ser285 747 stabilizes the inactive state of T2R4, and replacement of this residue with a non-polar amino acid, as in the case of S285A mutant, results in the receptor displaying a 3-5 fold increase in basal activity over wild type.
  • H214A Changes in this network brought about by mutations such as H214A cause the receptor to adopt an active conformation, and it involves the movement of TM6.
  • the H214A mutant showed constitutive activity of up to 10-fold over WT-T2R4, one of the highest reported for a GPCR mutant.
  • the intracellular region of T2R4 consists of 87 amino acids, including 4 alanines.
  • An N-terminal FLAG tagged T2R4 (WT-T2R4) was used as the base receptor and the entire intracellular region that includes 16 amino acids in ICL1 , 28 amino acids in ICL2, 23 amino acids in ICL3 and 16 amino acids in the C-terminus of T2R4 were replaced with alanines and the mutants pharmacologically characterized (Fig. 11 ).
  • the intensely bitter tasting natural alkaloid, quinine activates T2R4 in a concentration dependent manner.
  • the 83 alanine mutants of T2R4 displayed varied levels of cell surface expression, and calcium signaling upon stimulation with quinine (Table 2).
  • Majority of the 16 alanine mutants in ICL1 showed no significant changes in either cell surface expression and/or quinine induced changes in calcium mobilization.
  • Six ICL1 mutants showed defective agonist induced signaling.
  • the S51A, S52A, D53A and L56A mutants showed no detectable increase in intracellular calcium mobilization upon stimulation with quinine, while the F57A mutant displayed no concentration dependent increase in signal, and the signal was not saturated even at the highest quinine concentration.
  • the ICL2 in T2R4 has two triads of leucines, one in the center of the loop and the other at the end of the loop, at the ICL2-TM4 interface (Fig. 12).
  • the ICL2 alanine mutants only 15 of the 28 alanine replacements showed significant agonist induced signaling, with 7 of the mutants displaying defective ligand binding, as shown by more than a 1 .5-fold increase in EC50 mutant/wild type ratio (Table 2).
  • the C1 15A mutant in ICL2 showed the highest EC50 mutant/wild type ratio of 2.5 (Table 2).
  • the mutants L140A and L141A part of the second leucine triad in ICL2 were able to signal in response to quinine treatment, but not in a concentration dependent manner. Interestingly a majority of the mutants that were unable to signal were properly expressed at the cell surface. Compared to ICL2 mutants, the ICL3 mutants showed better agonist induced signaling with 14 of the 23 mutants displaying quinine induced signaling. Further, all of the mutants were targeted to the cell surface. Only three ICL3 mutants 1215A, F225A and P228A showed more than a 2-fold increase in EC 50 mutant/wild type ratio.
  • basal activity was calculated from slope of expression vs. basal activity for critical mutants compared to same value for the WT-T2R4. Therefore, all the 1 1 intracellular mutants that showed statistically significant increase in basal activity were expressed in HEK293T cells at different receptor densities, by varying the amount of DNA used in each transfection.
  • FIG. 1 shows a secondary structure representation of T2R4 amino acid sequence, and the amino acid sequence alignment of TM1 and TM7 of T2R1 , T2R4 and opsin.
  • T2R4 is found to be missing a residue at position 7.48 (Singh et al. 2011 a).
  • the residue at position 1.46 is a glycine in T2Rs
  • the amino acid at 7.47 is conserved as a serine in 70.2% or as proline in 18.1 % of the 188 T2Rs (Singh et al. 201 1a). in the remaining 11 % of the T2Rs, no amino acid conservation was observed at position 7.47.
  • the highly conserved residues in the two helices (Ballesteros and Weinstein residue) in both Class A GPCRs and T2Rs are also highlighted.
  • the natural alkaloid quinine and the synthetic bitter compound denatonium benzoate are agonists for T2R4. Both of these compounds are capable of stimulating T2R4 and cause a concentration-dependent increase in intracellular calcium in cells expressing T2R4 (Singh et al. 2011 b). Taste sensory analysis of both the bitter compounds using the analytical instrument E-TongueTM from Alpha MOS (Toulouse, France) showed that quinine has the most intense bitterness. Further, quinine, an important secondary metabolite, has higher efficacy for T2R4 and was thus selected for use as the agonist in the present study.
  • T2R4 receptors Functional analysis of wild type and mutant T2R4 receptors were determined by measuring changes in intracellular calcium of HEK293T cells transiently expressing these receptors after application of different concentrations of the T2R4 agonist quinine. All four mutants displayed varied levels of signaling ( Figure 2). The G28L and S285T mutants showed increased potency towards the agonist quinine, displayed as a left shift in dose-response. Among the five mutants, S285T displayed the highest potency with an EC50 value of 0.60 ⁇ 0.38 mM compared to 1.00 ⁇ 0.38 mM for WT-T2R4.
  • T2R4 Structure-function analysis on T2Rs revealed unique signature residues in TM helices that are distinct from Class A GPCRs (Singh et al., 2011 ; Pydi et al., 2012, J Neurochemistry 286: 36032-36041 ).
  • the intracellular region of T2R4 consists of 87 amino acids, including 4 alanines.
  • the natural alkaloid, quinine acts as an agonist and activates T2R4 in a concentration dependent manner.
  • the 23 alanine mutants in ICL3 of T2R4 displayed varied levels of calcium mobilization upon stimulation with quinine (Table 1 ). Only 14 of the 23 ICL3 alanine mutants displayed quinine induced signaling in a concentration dependent manner (Table 1 ). Three 1CL3 mutants, Q216A, T230A and V234A showed an increase in agonist induced response; however their response was not saturated even at the highest concentration of 5 mM quinine (Table 1 ).
  • Fetal Bovine Serum and DMEM High Glucose were purchased from Sigma and Invitrogen (Carlsbad, CA, USA). Common chemicals and bitter compounds were purchased either from Fisher or Sigma. Fluo-4NWTM and quinine hydrochloride, were purchased from Invitrogen and MP Biomedicals (Solon, OH, USA). All chemicals were of analytical grade and used without further purification.
  • HEK293T cells were cultured in 6-well tissue culture dishes at 37°C in DMEM-F12 and 10% FBS. Cells that are 70-80% confluent were co- transfected at 1 :1 ratio with T2R4 or mutants, and Ga 16/44 chimera (Ueda et ai.
  • Receptor activation was determined by measuring changes in intracellular calcium ( ⁇ Relative Fluorescence Units) after application of different concentrations of quinine or buffer alone (for measuring basal activity) using Flexstation-3TM fluorescence plate reader (Molecular Devices, CA, USA) at 525 nm following excitation at 494 nm. Calcium mobilized was expressed as ARFU after subtracting the responses of cells transfected with piasmid carrying the Ga16/44 construct, and in certain experiments was normalized to wild type cell surface expression as determined by ELISA. The data presented was from two to five independent transfections in triplicate. Dose-response curves were generated and ECso values calculated by non-linear regression analysis using PRISMTM software version 4.03 (GraphPad Software Inc, San Diego, CA).
  • the cells were then incubated with a concentration of 1 :5000 goat anti-mouse conjugated with horseradish peroxidase for 1 hour at room temperature, followed by washes as above. Empty cells were used as negative control. Color was developed by adding 200 ⁇ of SIGMAFASTTM OPD to each well by incubation in dark for 30 min, and absorbance was measured at 450 nm using Flexstation-3 fluorescence plate reader.
  • T2R4 amino acid sequence without the FLAG tag was used for model building, inactive and constitutively active T2R4 models were built using rhodopsin crystal structures, PDB ID: 11)19 and PDB ID: 2X72 respectively. Structural waters were introduced into these models using PyMol. Molecular dynamics simulations (10ns) were performed on all of the receptor models using SYBYL X1 .3 molecular modeling suite (Tripos Inc, USA).
  • Rhodopsin structure, dynamics, and activation a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. /Advances in protein chemistry, 63, 243-290.
  • Rasmussen, S. G., DeVree, B. T., Zou, Y. et al. (201 1) Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature, 477, 549-555.
  • Rhodopsin insights from recent structural studies. Annual review of biophysics and biomolecular structure, 31, 443-484.
  • Rhodopsin mutations in autosomal dominant retinitis pigmentosa Proceedings of the National Academy of Sciences of the United States of America, 88, 6481-6485.
  • T2R1 is activated by dipeptides and tripeptides. Biochemical and biophysical research communications, 398, 331-335. Table 1. Pharmacological characterization of the T2R4 alanine mutants. Functional characterization of the mutants was pursued by measuring intracellular calcium mobilized after stimulating with different concentrations of agonist, quinine. Cell surface expression was determined by ELISA*.
  • Intracellular loop 3 ICL3
  • ND- not detected no significant calcium mobilization detected
  • NS-not saturated quinine concentration dependent increase in calcium mobilization not observed.
  • Table 2 Pharmacological characterization of the T2R4 alanine mutants. Functional characterization of the mutants was pursued by measuring intracellular calcium mobilized after stimulating with different concentrations of a onist uinine. Cell surface ex re i *

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Abstract

Les récepteurs humains du goût amer (T2R) appartiennent à la superfamille des récepteurs couplés aux protéines G (GPCR). Les T2R partagent peu d'homologie avec la grande sous-famille de GPCR de Classe A, et leurs mécanismes d'activation ne sont guère compris. Guidés par des approches biochimiques et moléculaires, nous avons identifié deux acides aminés conservés Gly281.46 et Ser2857.47 présents sur les hélices transmembranaires (TM), TM1 et TM7, qui pourraient jouer des rôles importants dans l'activation des T2R. Nous avons muté Gly281.46 et Ser2857.47 dans T2R4 en G28A, G28L, S285A, S285T et S285P, et effectué une caractérisation pharmacologique des mutants. Le mutant S285A a présenté une activité agoniste-indépendante (~ 3 fois plus élevée que T2R4 de type sauvage basal ou S285T ou S285P). Ser2857.47 stabilise l'état inactif de T2R4 par un réseau de liaisons hydrogène reliant des restes importants sur TM1-TM2-TM7. A ce jour, S285A est le premier mutant de T2R constitutivement actif rapporté, et donne de nouvelles compréhensions dans l'activation de T2R.
PCT/CA2013/050313 2012-04-25 2013-04-23 Mutants constitutivement actifs des récepteurs du goût amer WO2013159227A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001018050A2 (fr) * 1999-09-10 2001-03-15 The Regents Of The University Of California T2r, nouvelle famille de recepteurs du gout
US7776561B2 (en) * 2001-07-10 2010-08-17 Senomyx, Inc. Use of specific T2R receptors to identify compounds that block bitter taste

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001018050A2 (fr) * 1999-09-10 2001-03-15 The Regents Of The University Of California T2r, nouvelle famille de recepteurs du gout
US7776561B2 (en) * 2001-07-10 2010-08-17 Senomyx, Inc. Use of specific T2R receptors to identify compounds that block bitter taste

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PYDI SP ET AL.: "Consitutively active mutant gives novel insights into the mechanism of bitter taste receptor activation.", J. OF NEUROCHEMISTRY, vol. 122, August 2012 (2012-08-01), pages 537 - 544 *

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